Composition and method for tumor imaging

ABSTRACT

A method is provided for enhancing transmembrane transport of a diagnostic agent across a membrane of a living cell. The method comprises contacting a membrane of a living cell with a complex formed between said diagnostic agent and ligands selected from biotin or biotin receptor-binding analogs of biotin, folate or folate receptor-binding analogs of folate, riboflavin or riboflavin receptor-binding analogs of riboflavin to initiate receptor mediated transmembrane transport of the ligand complex. The method is used for imaging tissues in vivo.

This invention was made with Government support under Grant89-45-DCB-88-11465, awarded by the National Science Foundation and GrantR01-CA46909 awarded by the National Cancer Institute. The Government hascertain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/349,407, filed Dec. 5, 1994, which is a continuation of U.S.application Ser. No. 07/851,544, filed Mar. 13, 1992, issued as U.S.Pat. No. 5,416,016, which is a continuation of U.S. application Ser. No.07/498,762, filed Mar. 28, 1990, issued as U.S. Pat. No. 5,108,921,which is a continuation-in-part of U.S. application Ser. No. 07/331,816,filed Apr. 3, 1989, now abandoned.

FIELD OF THE INVENTION

This invention relates to a method for enhancing transmembrane transportof exogenous molecules. More particularly, the use of nutrientreceptors, including biotin or folate receptors, and the respectiveassociated receptor mediated endocytotic mechanism associated with suchreceptors, is utilized to enhance the efficiency of cellular uptake ofexogenous molecules capable of modulating or otherwise modifying cellfunction.

BACKGROUND AND SUMMARY OF THE INVENTION

Transmembrane transport of nutrient molecules is a critical cellularfunction. Because practitioners have recognized the importance oftransmembrane transport to many areas of medical and biological science,including drug therapy and gene transfer, there has been significantresearch efforts directed to the understanding and application of suchprocesses. Thus, for example, transmembrane delivery of nucleic acidshas been encouraged through the use of protein carriers, antibodycarriers, liposomal delivery systems, electroporation, direct injection,cell fusion, viral carriers, osmotic shock, and calcium-phosphatemediated transformation. However, many of those techniques are limitedboth by the types of cells in which transmembrane transport is enabledand by the conditions of use for successful transmembrane transport ofexogenous molecular species. Further, many of these known techniques arelimited in the type and size of exogenous molecule that can betransported across a membrane without loss of bioactivity.

One method for transmembrane delivery of exogenous molecules having awide applicability is based on the mechanism of receptor mediatedendocytotic activity. Unlike many other methods, receptor mediatedendocytotic activity can be used successfully both in vivo and in vitro.Receptor mediated endocytosis involves the movement of ligands bound tomembrane receptors into the interior of an area bounded by the membranethrough invagination of the membrane. The process is initiated oractivated by the binding of a receptor specific ligand to the receptor.Many receptor mediated endocytotic systems have been characterized,including those recognizing galactose, mannose, mannose 6-phosphate,transferrin, asialoglycoprotein, transcobalamin (vitamin B₁₂), α-2macroglobulins, insulin, and other peptide growth factors such asepidermal growth factor (EGF).

Receptor mediated endocytotic activity has been utilized for deliveringexogenous molecules such as proteins and nucleic acids to cells.Generally, a specified ligand is chemically conjugated by covalent,ionic or hydrogen bonding to an exogenous molecule of interest, (i.e.,the exogenous compound) forming a conjugate molecule having a moiety(the ligand portion) that is still recognized in the conjugate by atarget receptor. Using this technique the phototoxic protein psoralenhas been conjugated to insulin and internalized by the insulin receptorendocytotic pathway (Gasparro, Biochem. Biophys. Res. Comm. 141(2), pp.502-509, Dec. 15, 1986); the hepatocyte specific receptor for galactoseterminal asialoglycoproteins has been utilized for thehepatocyte-specific transmembrane delivery ofasialoorosomucoid-poly-L-lysine non-covalently complexed to a DNAplasmid (Wu, G. Y., J. Biol. Chem., 262(10), pp. 4429-4432, 1987); thecell receptor for epidermal growth factor has been utilized to deliverpolynucleotides covalently linked to EGF to the cell interior (Myers,European Patent Application 86810614.7, published Jun. 6, 1988); theintestinally situated cellular receptor for the organometallic vitaminB₁₂ -intrinsic factor complex has been used to mediate delivery to thecirculatory system of a vertebrate host a drug, hormone, bioactivepeptide or immunogen complexed with vitamin B₁₂ and delivered to theintestine through oral administration (Russell-Jones et al., Europeanpatent Application 86307849.9, published Apr. 29, 1987); themannose-6-phosphate receptor has been used to deliver low densitylipoproteins to cells (Murray, G. J. and Neville, D. M., Jr., J. Bio.Chem, Vol. 255 (24), pp. 1194-11948, 1980); the cholera toxin bindingsubunit receptor has been used to deliver insulin to cells lackinginsulin receptors (Roth and Maddox, J. Cell. Phys. Vol. 115, p. 151,1983); and the human chorionic gonadotropin receptor has been employedto deliver a ricin a-chain coupled to HCG to cells with the appropriateHCG receptor in order to kill the cells (Oeltmann and Heath, J. Biol.Chem, vol. 254, p. 1028 (1979)).

The method of the present invention enhances the transmembrane transportof an exogenous molecule across a membrane having biotin or folatereceptors that initiate transmembrane transport of receptor boundspecies. The method takes advantage of (1) the location and multiplicityof biotin and folate receptors on the membrane surfaces of most cellsand (2) the associated receptor mediated transmembrane processes.Performance of the method involves formation of a complex between aligand selected from biotin or other biotin receptor-binding compounds,and/or folic acid or other folate receptor-binding compounds, and anexogenous molecule. A cell membrane bearing biotin or folate receptorsis contacted with this complex, thereby initiating receptor mediatedtransmembrane transport of the complex. The complex is allowed tocontact the membrane surface bearing the corresponding receptors for atime sufficient to initiate and permit transmembrane transport of thecomplex. The transmembrane transport of exogenous molecules includingproteins and polynucleotides has been promoted in plant, mammalian, andbacterial cells.

In one embodiment of this invention, the target receptor for the methodof the present invention is the biotin receptor. Biotin is a necessarycellular nutrient that has been found to be preferentially bound bybiotin receptor proteins associated with cellular membranes.Commercially available reagents are used to form a covalent complexbetween biotin and polynucleotides, proteins, or other desired exogenousmolecules. According to one preferred embodiment of the presentinvention, a biotin/exogenous molecule complex is brought into contactwith a membrane having associated biotin receptors for a time sufficientto allow binding of the biotin moiety of the complex to a correspondingbiotin receptor in the membrane. This binding triggers the initiation ofcellular processes that result in transmembrane transport of thecomplex.

In an alternate but equally preferred embodiment of this invention,folate receptors are targeted to enhance cellular uptake of exogenousmolecules. Folate binding receptors are found in most types of cells,and they have been demonstrated to bind and trigger cellularinternalization of folates. Thus, folic acid and other art-recognizedfolate receptor-binding ligands can be chemically bonded topolynucleotides, proteins, or other desired exogenous molecules usingart-recognized coupling techniques to provide a folate receptor-bindingcomplex which is readily endocytosed into living cells. In accordancewith this embodiment of the present invention, a folate/exogenousmolecule complex is brought into contact with a membrane havingassociated folate receptors for a time sufficient to allow binding ofthe folate moiety of the complex to a corresponding folate receptor.Folate receptor-binding triggers the initiation of cellular processesthat result in transmembrane transport of the complex.

The methods of this invention are particularly useful for increasing theinternalization efficiency (cellular uptake) of exogenous molecules thatare normally resistant to cellular internalization. Proteins andpolynucleotides previously recognized as difficult to move across cellmembranes can be internalized by a cell through application of themethod of the present invention. For example, transformation of targetcell lines resulting in expression of a protein product has beenaccomplished by coupling the desired polynucleotide to either biotin orfolates, and contacting the cells with the resulting complex for a timesufficient to promote cellular internalization. In one case, a DNAplasmid containing a gene sequence coding for chloramphenicolacetyltransferase (CAT), was biotinylated and transported into E. colivia a biotin receptor mediated endocytotic pathway and expressed.Similar examples of transformation or transection have been noted forbiotin or folate linked nucleic acids in mammalian systems, prokaryoticsystems, and plants. The use of biotin and folate complexes to enhancecellular uptake of complexed exogenous molecules has been demonstratedin vivo and in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structures of chelators useful for forming thefolate-radionuclide complexes of the present invention.

FIG. 2 illustrates the measured mouse serum folate levels as a functionof time following initiation of the folate-deficient diet.

FIG. 3 is an illustration of a deferoxamine-folate conjugate which canbe radiolabeled with ⁶⁷ Ga.

FIG. 4 is a graphic representation of the cellular uptake by BHK cellsof ¹²⁵ I labeled ribonuclease/riboflavin conjugates. At varyingtimepoints, the cells were washed 5× in saline, and counted in a gammacounter.

FIG. 5 illustrates the biodistribution of ¹²⁵ I-BSA-riboflavin conjugatefollowing administration to Wistar female rats. The solid bars representthe bovine serum albumin (BSA) content of tissues from rats treated withthe ¹²⁵ I-BSA-riboflavin conjugated samples, while the open barsrepresent the bovine serum albumin (BSA) content of tissues from ratstreated with ¹²⁵ I-BSA.

FIG. 6 illustrates the cellular internalization of thiamin-BSA andriboflavin-BSA complexes by cultured A549 cells.

FIG. 7 is a graphic representation of the time dependant uptake of BSAand BSA-thiamin complexes by KB cells.

FIG. 8 illustrates the percent injected dose of ⁶⁷ Ga-radiotracer (⁶⁷Ga-citrate, ⁶⁷ Ga-deferoxamine, and ⁶⁷ Ga-deferoxamine-folate) per gramtumor. Each bar represents the data from one animal. Group 1 wasadministered ⁶⁷ Ga-deferoxamine-folate; Group 2 was administered ⁶⁷Ga-deferoxamine-folate to mice maintained on a high folate diet; Group 3was administered folic acid (approximately 2.4 mg) prior toadministration of ⁶⁷ Ga-deferoxamine-folate; Group 4 was administered ⁶⁷Ga-deferoxamine-folate with a chase dose of folate one hour prior tosacrifice; Group 5 was administered ⁶⁷ Ga-deferoxamine; Group 6 wasadministered ⁶⁷ Ga-citrate.

FIG. 9 illustrates the tumor to blood ratios (% of injected dose pergram wet weight) at 4-4.5 hours post-injection for ⁶⁷ Ga-radiotracers:⁶⁷ Ga-citrate, ⁶⁷ Ga-deferoxamine, and ⁶⁷ Ga-deferoxamine-folate. Eachbar represents data from one animal. Group 1 was administered ⁶⁷Ga-deferoxamine-folate; Group 2 was administered ⁶⁷Ga-deferoxamine-folate to mice maintained on a high folate diet; Group 3was administered folic acid (approximately 2.4 mg) prior toadministration of ⁶⁷ Ga-deferoxamine-folate; Group 4 was administered ⁶⁷Ga-deferoxamine-folate with a chase dose of folate one hour prior tosacrifice; Group 5 was administered ⁶⁷ Ga-deferoxamine; Group 6 wasadministered ⁶⁷ Ga-citrate.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one embodiment of this invention, there is provided amethod for enhancing transport of an exogenous molecule across amembrane of a living cell. The method comprises the step of contactingthe membrane with the exogenous molecule complexed with a ligandselected from the group consisting of biotin, biotin receptor-bindinganalogs of biotin, and other biotin receptor-binding ligands, for a timesufficient to permit transmembrane transport of said ligand complex. Ina second embodiment, there is provided a method for enhancing transportof an exogenous molecule across a membrane of a living cell, comprisingthe step of contacting the membrane with the exogenous moleculecomplexed with a ligand selected from the group consisting of folicacid, folate receptor-binding analogs of folic acid, and other folatereceptor-binding ligands, for a time sufficient to permit transmembranetransport of said ligand complex.

The method of the present invention is effective in all living cellsthat have biotin and/or folate receptors associated with their cellularmembranes. The membrane can define an intracellular volume such as theendoplasmic reticulum or other organelles such as mitochondria, oralternatively, the membrane can define the boundary of the cell.

Living cells which can serve as the target for the method of thisinvention include prokaryotes and eukaryotes, including yeasts, plantcells and animal cells. The present method can be used to modifycellular function of living cells in vitro, i.e., in cell culture, or invivo, where the cells form part of or otherwise exist in plant tissue oranimal tissue. Thus the cells can form, for example, the roots, stalksor leaves of growing plants and the present method can be performed onsuch plant cells in any manner which promotes contact of the exogenousmolecule/folate or biotin complex with the targeted cells having therequisite receptors. Alternatively, the target cells can form part ofthe tissue in an animal. Thus the target cells can include, for example,the cells lining the alimentary canal, such as the oral and pharyngealmucosa, the cells forming the villi of the small intestine, or the cellslining the large intestine. Such cells of the alimentary canal can betargeted in accordance with this invention by oral administration of acomposition comprising an exogenous molecule complexed with folates orbiotin or their receptor-binding analogs. Similarly, cells lining therespiratory system (nasal passages/lungs) of an animal can be targetedby inhalation of the present complexes; dermal/epidermal cells and cellsof the vagina and rectum can be targeted by topical application of thepresent complexes; and cells of internal organs including cells of theplacenta and the so-called blood/brain barrier can be targetedparticularly by parenteral administration of the present complexes.Pharmaceutical formulations for therapeutic use in accordance with thisinvention containing effective amounts of the presently described folateand biotin complexes, in admixture with art-recognized excipientsappropriate to the contemplated route of administration are within thescope of this invention.

Since not all natural cell membranes possess biologically active biotinor folate receptors, practice of the method of this invention in vitroon a particular cell line can involve altering or otherwise modifyingthat cell line first to ensure the presence of biologically activebiotin or folate receptors. Thus, the number of biotin or folatereceptors on a cell membrane can be increased by growing a cell line onbiotin or folate deficient substrates to promote biotin and folatereceptor production, or by expression of an inserted foreign gene forthe protein or apoprotein corresponding to the biotin or folatereceptor.

The present invention is utilized to enhance the cellular uptake ofexogenous molecules, in particular those molecules capable of modulatingor otherwise modifying cell function, including pharmaceutically activecompounds or diagnostic agents. Suitable exogenous molecules caninclude, but are not limited to: peptides, oligopeptides, proteins,apoproteins, glycoproteins, antigens and antibodies thereto, haptens andantibodies thereto, receptors and other membrane proteins, retro-inversooligopeptides, protein analogs in which at least one non-peptide linkagereplaces a peptide linkage, enzymes, coenzymes, enzyme inhibitors, aminoacids and their derivatives, hormones, lipids, phospholipids, liposomes;toxins such as aflatoxin, digoxin, xanthotoxin, rubratoxin; antibioticssuch as cephalosporins, penicillin, and erythromycin; analgesics such asaspirin, ibuprofen, and acetaminophen, bronchodilators such theophyllineand albuterol; beta-blockers such as propranolol, metoprolol, atenolol,labetolol, timolol, penbutolol, and pindolol; antimicrobial agents suchas those described above and ciprofloxacin, cinoxacin, and norfloxacin;antihypertensive agents such as clonidine, methyldopa, prazosin,verapamil, nifedipine, captopril, and enalapril; cardiovascular agentsincluding antiarrhythmics, cardiac glycosides, antianginals andvasodilators; central nervous system agents including stimulants,psychotropics, antimanics, and depressants; antiviral agents;antihistamines such as chlorpheniramine and brompheniramine; cancerdrugs including chemotherapeutic agents; tranquilizers such as diazepam,chordiazepoxide, oxazepam, alprazolam, and triazolam; anti-depressantssuch as fluoxetine, amitriptyline, nortriptyline, and imipramine; H-2antagonists such as nizatidine, cimetidine, famotidine, and ranitidine;anticonvulsants; antinauseants; prostaglandins; muscle relaxants;anti-inflammatory substances;; stimulants; decongestants; antiemetics;diuretics; antispasmodics; antiasthmatics; anti-Parkinson agents;expectorants; cough suppressants; mucolytics; vitamins; and mineral andnutritional additives. Other molecules include nucleotides;oligonucleotides; polynucleotides; and their art-recognized andbiologically functional analogs and derivatives including, for example;methylated polynucleotides and nucleotide analogs havingphosphorothioate linkages; plasmids, cosmids, artificial chromosomes,other nucleic acid vectors; antisense polynucleotides including thosesubstantially complementary to at least one endogenous nucleic acid orthose having sequences with a sense opposed to at least portions ofselected viral or retroviral genomes; promoters; enhancers; inhibitors;other ligands for regulating gene transcription and translation, and anyother biologically active molecule that cad form a complex with biotinor folate, or analogs thereof, by direct conjugation of the exogenousmolecule with biotin or biotin analog or folate or folate analog througha hydrogen, ionic, or covalent bonding. Also in accordance with thisinvention is the use of indirect means for associating the exogenousmolecule with biotin or folate, or analogs thereof to form liquidcomplexes, such as by connection through intermediary linkers, spacerarms, bridging molecules, or liposome entrapment, all of which can actto associate the biotin or biotin analog or folate or folate analog withthe exogenous molecule of interest. Both direct and indirect means forassociating the ligand and the exogenous molecule must not prevent thebinding of the ligand held in association with the exogenous molecule toits respective ligand receptor on the cell membrane for operation of themethod of the present invention.

Generally, any manner of forming a complex between an exogenous moleculeof interest and a ligand capable of triggering receptor mediatedendocytosis can be utilized in accordance with the present invention.This can include covalent, ionic, or hydrogen bonding of the ligand tothe exogenous molecule, either directly or indirectly via a linkinggroup. The complex is typically formed by covalent bonding of thereceptor-activating moiety to the exogenous molecule through theformation of amide, ester or imino bonds between acid, aldehyde,hydroxy, amino, or hydrazo groups on the respective components of thecomplex. Art-recognized biologically labile covalent linkages such asimino bonds (--C═N--) and so-called "active" esters having the linkage--COOCH₂ O or --COOCH(CH₃)O are preferred, especially where theexogenous molecule is found to have reduced functionality in thecomplexed form. Hydrogen bonding, e.g., that occurring betweencomplementary strands of nucleic acids, can also be used for complexformation. Thus a biotinylated or folated oligonucleotide complementaryto at least a portion of a nucleic acid to be delivered to a cell inaccordance with this invention can be hybridized with said nucleic acidand the hybrid (complex) used per this invention to enhance delivery ofthe nucleic acid into cells.

Because of the ready availability of biotinylating reagents andbiotinylating methods suitable for use with peptides, proteins,oligonucleotides, polynucleotides, lipids, phospholipids, carbohydrates,liposomes or other lipid vesicles, lower molecular weight therapeuticagents, bioactive compounds, and carriers for therapeutic agents, biotinis a preferred complex forming ligand for use in carrying out thisinvention. Generally, the biotin/exogenous molecule complex is formed bycovalently binding biotin or a biotin derivative to the exogenousmolecule of interest. Transmembrane transport via the biotin/biotinreceptor pathway is also preferred because biotin is a necessarynutrient for a wide variety of cells, and biotin receptors that mediateendocytotic activity have been identified in mammalian, plant, andbacterial cells.

Formation of a complex between biotin and an exogenous molecule ofinterest is readily accomplished. Biotin and its analogs can be easilyconjugated to proteins by activating the carboxyl group of biotin,thereby making it reactive with the free amino groups of the proteins toform a covalent amide linking bond. A biotinylating reagent such asD-biotin-N-hydroxy-succinimide ester or biotinyl-p-nitrophenyl ester canbe used. The activated ester reacts under mild conditions with aminogroups to incorporate a biotin residue into the desired molecule. Theprocedure to be followed for biotinylating macromolecules usingD-biotin-N-hydroxy-succinimide ester is well known in the art (Hofmannet al., J. Am. Chem. Soc. 100, 3585-3590 (1978)). Procedures suitablefor biotinylating an exogenous molecule using biotinyl-p-nitrophenylester as a biotinylating reagent are also well known in the art(Bodanszk et al., J. Am. Chem. Soc. 99, 235 (1977)). Other reagents suchas D-biotinyl-ε-aminocaproic acid N-hydroxy-succinimide ester in whichε-aminocaproic acid serves as a spacer link to reduce steric hindrancecan also be used for the purposes of the present invention.

Oligonucleotides and polynucleotides can also be biotinylated using bothindirect and direct methods. Indirect methods include end-labeling of apolynucleotide with a biotinylated nucleotide, or nick translation thatincorporates biotinylated nucleotides. Nick translation or end labelingof DNA can be accomplished using methods described in Maniatis et al.,Molecular Cloning: A Laboratory Manual, pp. 109-116, Cold Spring HarborPress (1982). Direct methods are those procedures in which biotin isdirectly attached to a target polynucleotide using a biotinylatingreagent. Photoactivatible reagents such as the acetate salt ofN-(4-azido-2-nitrophenyl)-N-(3-biotinylaminopropyl)-N-methyl-1,3-propanediamine(photobiotin) can be used to biotinylate DNA according to the method ofForster et al., Nuc. Acids Res. 13:745-761. An alternative method uses abiotin hyrazide reagent in a bisulfite catalyzed reaction capable oftransamination of nucleotide bases such as cytidine according to themethod described by Reisfeld et al., B.B.R.C. 142:519-526 (1988). Thismethod simply requires a 24 hour incubation of DNA or RNA with biotinhydeazide at 10mg/ml in an acetate buffer, pH 4.5, containing 1Mbisulfite. Biotin hydrazide can also be used to biotinylatecarbohydrates or other exogenous molecules containing a free aldehyde.

Biotin analogs such as biocytin, biotin sulfoxide, oxybiotin and otherbiotin receptor-binding compounds are liquids that may also be used assuitable complexing agents to promote the transmembrane transport ofexogenous molecules in accordance with this invention. Other compoundscapable of binding to biotin receptors to initiate receptor mediatedendocytotic transport of the complex are also contemplated. Such caninclude other receptor-binding ligands such as, for example,anti-idiotypic antibodies to the biotin receptor. An exogenous moleculecomplexed with an anti-idiotypic antibody to a biotin receptor could beused to trigger transmembrane transport of the complex in accordancewith the present invention.

Folate receptors that mediate endocytotic activity have previously beenidentified in bacterial cells (Kumar et al., J. Biol. Chem., 262,7171-79 (1987)). Folic acid, folinic acid, pteropolyglutamic acid, andfolate receptor-binding pteridines such as tetrahydropterins,dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogsare preferred complex-forming ligands used in accordance with a secondembodiment of this invention. The terms "deaza" and "dideaza" analogsrefers to the art recognized analogs having a carbon atom substitutedfor one or two nitrogen atoms in the naturally occurring folic acidstructure. For example, the deaza analogs include the 1-deaza, 3-deaza,5-deaza, 8-deaza, and 10-deaza analogs. The dideaza analogs include, forexample, 1,5 dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideazaanalogs. The foregoing folic acid derivatives are conventionally termed"folates", reflecting their capacity to bind with folate-receptors, andsuch ligands when complexed with exogenous molecules are effective toenhance transmembrane transport. Other folates useful as complex formingligands for this invention are the folate receptor-binding analogsaminopterin, amethopterin (methotrexate), N¹⁰ -methylfolate,2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin or3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid (dichloromethotrexate). Other suitableligands capable of binding to folate receptors to initiate receptormediated endocytotic transport of the complex include anti-idiotypicantibodies to the folate receptor. An exogenous molecule in complex withan anti-idiotypic antibody to a folate receptor is used to triggertransmembrane transport of the complex in accordance with the presentinvention.

Folated ligands can be complexed with the exogenous moleculeshereinbefore defined using art-recognized covalent coupling techniquesidentical to or closely paralleling those referenced above for thebiotinylate ligand complexes. Thus, for example, a carboxylic acid onthe folate moiety or on the exogenous molecule can be activated using,for example, carbonyldiimidazole or standard carbodimide couplingreagents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andthereafter reacted with the other component of the complex having atleast one nucleophilic group, viz hydroxy, amino, hydrazo, or thiol, toform the respective complex coupled through an ester, amide, orthioester bond. Thus complexes can be readily formed between folateligands and peptides, proteins, nucleic acids, including both RNA andDNA, phoshorodithioate analogs of nucleic acids, oligonucleotides,polynucleotides, lipids and lipid vesicles, phospholipids, carbohydratesand like exogenous molecules capable of modifying cell function. Theligand complexes enable rapid, efficient delivery of the cellfunction-modifying moiety through cellular membranes and into the cell.

It is contemplated that both folate and biotinylate-receptor bindingligands can be used advantageously in combination to deliver exogenousmolecules through cell membranes. Thus, for example, an exogenousmolecule can be multiply conjugated with both folate and biotinylateligands to enhance opportunity for binding with the respective cellmembrane receptors. Alternatively, independent portions of a dose of anexogenous compound can be biotinylated and folate-coupled, respectively,and the portions of the resulting complexes can subsequently be combinedto provide a mixture of ligand complexes for modification of cellfunction.

Receptor mediated cellular uptake of biotinylated or folate-derivatizedpolynucleotides provides a convenient, efficient mechanism fortransformation of cells. The method is particularly valuable for celltransformation because it is applicable even to cell types, such asplant cells, which are normally resistant to standard transformationtechniques. Delivery of foreign genes to The cell cytoplasm can beaccomplished with high efficiency using the present invention. Oncedelivered through the cell membrane to the cell interior, foreign genescan be expressed to produce a desired protein. In addition, othernucleic acids can be introduced, for example, an antisense-RNA sequencecapable of binding interference with endogenous messenger RNA.

Artificially generated phospholipid vesicles have been used as carriersfor introducing membrane-impermeable substancas into cells, asinstruments for altering lipid composition of membranes in intact cells,and as inducers of cell fusion. Liposome/cell membrane interaction ispotentiated in accordance with one application of the method of thisinvention by contacting the cell membrane with a liposome containing theexogenous molecule and bearing ligands on its membrane contactingsurface. For example, liposome-forming phospholipids can be biotinylatedor folate-conjugated through, for example, headgroup functional groupssuch as hydroxy and amino groups. The resulting phospholipid/ligandcomplex is then used itself or in combination with unmodifiedphospholipids to form liposomes containing exogenous molecules capableof modulating or otherwise modifying cell function. The resultingliposomes, again formed in whole or in part from the phospholipid/biotinor folate complex, present biotin or folate receptor-binding groups tothe cell surface, triggering the receptor mediated endocytosismechanism, thereby promoting delivery of the liposome-containedsubstances into the cell. One readily available phospholipid that can beused in accordance with the above-described method isphosphatidylethanolamine. That phosphollpid can be convenientlycomplexed using art-recognized procedures with either biotin, biotinanalogs or folate-receptor-binding ligands to form a phospholipid/ligandcomplex. The receptor-binding complex can be combined with otherphospholipids, for example, phosphatidylcholine and that mixture can beused to form liposomes containing biologically active substances fordelivery of those biologically active substances to cells.

It is further contemplated in accordance with this invention that othercell nutrients for which there exists receptors and associated receptormediated endocytotic uptake could serve as ligands for forming complexeswith exogenous molecules to enhance their cellular uptake. Amongnutrients believed to trigger receptor mediated endocytosis and havingapplication in accordance with the presently disclosed method arecarnitine, inositol, lipoic acid, niacin, pantothenic acid, riboflavln,thiamin, pyridoxal, and ascorbic acid, and the lipid soluble vitamins A,D, E and K. These non-organometallic nutrients, and their analogs andderivatives thereof, constitute ligands that can be coupled withexogenous molecules to form ligand complexes for contact with cellmembranes following the same procedures described hereinabove for biotinand folate. These foregoing nutrients are generally required nutrientsfor mammalian cells. Exogenous molecules coupled with the foregoingnon-organometallic nutrients can be used to deliver effective amounts oftherapeutic agents or pharmaceutically active agents such as previouslydescribed through parenteral or oral routes of administration to humanor animal hosts.

In accordance with one embodiment of the present invention, theexogenous molecule comprises a diagnostic agent that is complexed with aligand to enhance transport of the diagnostic agent across a membrane ofa living cell. The ligand is selected from the group consisting ofbiotin or biotin receptor-binding analogs of biotin, folate or folatereceptor-binding analogs of folate, riboflavin or riboflavinreceptor-binding analogs of riboflavin, and thiamin or thiaminreceptor-binding analogs of thiamin. Complexing a vitamin ligand to adiagnostic agent allows the diagnostic agent to be targeted, uponadministration to an animal, to tissues that possess membrane-boundreceptors for the vitamin ligand. This results in an enhancedconcentration of the diagnostic agent at the target tissues and providesrapid clearance of the diagnostic agent from non-target tissue.

Diagnostic agents suitable for use in the present invention include anycompound that is capable of being detected in vivo after administrationto a multicellular organism. Preferred compounds include electron densematerials, magnetic resonance imaging agents and radiopharmaceuticals.

The ligand can be complexed to the diagnostic agent by covalent, ionicor hydrogen bonding either directly or indirectly through a linkinggroup. In one embodiment the diagnostic agent is contained in aliposome, wherein the liposome comprises liposome-forming phospholipds,at least a portion of which are covalently bound through theirheadgroups to the ligand.

In one embodiment ligands selected from the group consisting of biotinor biotin receptor-binding analogs of biotin, folate or folatereceptor-binding analogs of folate, riboflavin or riboflavinreceptor-binding analogs of riboflavin, and thiamin or thiaminreceptor-binding analogs of thiamin, are coupled to radionuclides andused for diagnostic imaging. Radionuclide suitable for diagnosticimaging include radioisotopes of gallium, indium, copper, technetium andrhenium, including isotopes ¹¹¹ In, ^(99m) Tc, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga or ⁶⁸Ga. These radionuclides can be conjugated to a vitamin ligand through achelating linking group. The chemical structure of the chelating agentis not critical provided that it have the requisite affinity for theradionuclide cation. Suitable chelating agents for use in accordancewith the present invention include the chelates shown in FIG. 1 as wellas tetraazacyclotetradecanetetraacetate (TETA).

In one embodiment of the present invention a ligand-radiopharmaceuticalcomplex is used to image tumor cells. In particular, folicacid-radionuclide complexes have been used to image tumor cells. Folicacid is an essential dietary vitamin needed by all eucaryotic cells forDNA synthesis and carbon metabolism. Folic acid primarily enters cellsthrough facilitated transport by a membrane transport protein (K_(m)=1.5×10⁻⁶ M for folic acid), however, some cells also possess amembrane-bound folate-binding-protein receptor (FBP) that secondarilyallows folate uptake via receptor mediated endocytosis (K_(a) =5×10⁻¹⁰ Mfor folate). When folate is covalently bonded, directly or indirectlythrough a linking group, to a diagnostic agent via itsgamma-carboxylate, the folate fragment ceases to be recognized by thefacilitated transport system, but can still be recognized by the FBPreceptor. Thus, such folate-conjugates are selectively concentrated bycells that express the membrane FBP receptor.

A number of tumor cell types (e.g. breast, ovarian, cervical,colorectal, renal, and nasopharyngeal) are known to overexpress FBPreceptors. Conjugation of diagnostic agents, such asradiopharmaceuticals, to the gamma-carboxylate of folate enhances theselective uptake of these complexes by tumor cells allowing for morerapid and sensitive imaging of tumors.

¹²⁵ I labeled ribonuclease-folate was used to evaluate radiotracerdelivery to tumor cells in athymic mice maintained on a folate-free diet(to regulate serum folate concentration closer to levels found in normalhuman serum). Tumor cells were implanted in athymic mice by subcutaneousinjection of 2×10⁶ human KB cells into the shoulder of the mice. Themice were administered the ¹²⁵ I labeled ribonuclease-folate conjugateintravenously via the femoral vein twenty days after subcutaneousinjection the human KB cells. As a control, tumor bearing athymic micewere injected with ¹²⁵ I labeled ribonuclease (lacking folate). Thebiodistribution of each agent, calculated as a percentage of theinjected dose per gram of tissue, is shown in tables 1 (¹²⁵I-ribonuclease-folate) and 2 (¹²⁵ I-ribonuclease-folate). Some tumorselectively is apparent based on comparison of the tumor uptake andtumor/blood ratios for ¹²⁵ I-ribonuclease-folate and ¹²⁵ I-ribonuclease.However, this level of selectivity is not sufficient to afford clinicalutility, due to poor tumor contrast with other non-target tissues.

                  TABLE 1                                                         ______________________________________                                        Biodistribution of .sup.125 I-RNase-Folate Conjugate                          Following I.V. Administration to Male Athymic                                 Mice (Folate-free diet) With KB Tumors                                               Percentage of Injected Dose per Gram                                          1 hour    4 hours     24 hours                                         ______________________________________                                        Blood    5.34 ± 1.07                                                                            2.30 ± 0.62                                                                            0.04 ± 0.01                               Heart    2.01 ± 0.22                                                                            0.93 ± 0.39                                                                            0.024 ± 0.006                             Lungs    4.19 ± 0.61                                                                            1.97 ± 0.77                                                                            0.04 ± 0.01                               Liver    9.78 ± 1.11                                                                            3.29 ± 0.77                                                                            0.38 ± 0.03                               Spleen   9.94 ± 1.37                                                                            2.78 ± 0.73                                                                            0.23 ± 0.04                               Kidney   16.08 ± 2.76                                                                           5.11 ± 1.27                                                                            0.72 ± 0.07                               Brain    0.29 ± 0.04                                                                            0.19 ± 0.14                                                                            0.007 ± 0.001                             Muscle   1.73 ± 0.32                                                                            0.98 ± 0.54                                                                            0.013 ± 0.002                             Testes   1.50 ± 0.18                                                                            *1.0 ± 0.15                                                                            0.021 ± 0.004                             Bone     3.20 ± 0.23                                                                            1.17 ± 0.28                                                                            0.09 ± 0.03                               Thyroid  --          --          --                                           Tumor    5.35 ± 0.54                                                                            2.74 ± 0.51                                                                            0.41 ± 0.02                               Stomach  21.07 ± 2.57                                                                           26.45 ± 8.05                                                                           0.26 ± 0.14                               Intestines                                                                             2.03 ± 0.28                                                                            0.95 ± 0.16                                                                             0.05 ± 0.006                             Tumor/Blood                                                                            1.02 ± 0.16                                                                            1.21 ± 0.17                                                                            11.88 ± 3.71                              Tumor/Muscle                                                                           3.14 ± 0.43                                                                            3.35 ± 1.55                                                                            32.6 ± 6.7                                n        4           3           3                                            ______________________________________                                         *n = 2                                                                   

                  TABLE 2                                                         ______________________________________                                        Biodistribution of .sup.125 I-RNase (Control) Following                       I.V. Administration to Male Athymic                                           Mice (Folate-free diet) With KB Tumors                                               Percentage of Injected Dose per Gram                                          1 hour    4 hours     24 hours                                         ______________________________________                                        Blood    4.99 ± 1.22                                                                            1.06 ± 7.31                                                                            0.06 ± 0.01                               Heart    1.72 ± 0.41                                                                            0.40 ± 0.09                                                                            0.026 ± 0.007                             Lungs    3.65 ± 0.81                                                                            0.79 ± 0.21                                                                            0.047 ± 0.012                             Liver    1.83 ± 0.72                                                                            0.42 ± 0.13                                                                            0.91 ± 1.73                               Spleen   2.12 ± 0.50                                                                            0.53 ± 0.14                                                                            0.023 ± 0.006                             Kidney   24.3 ± 5.4                                                                             5.84 ± 0.18                                                                            1.66 ± 0.26                               Brain    0.19 ± 0.06                                                                            0.06 ± 0.004                                                                           0.0055 ± 0.0006                           Muscle   1.27 ± 0.24                                                                            0.35 ± 0.10                                                                            0.021 ± 0.005                             Testes   1.79 ± 1.18                                                                            0.52 ± 0.05                                                                            0.017 ± 0.005                             Bone     1.89 ± 0.31                                                                            0.55 ± 0.16                                                                            0.07 ± 0.05                               Thyroid  --          --          --                                           Tumor    3.31 ± 1.75                                                                            0.87 ± 0.26                                                                            0.038 ± 0.012                             Stomach  21.37 ± 9.72                                                                           9.80 ± 4.24                                                                            0.21 ± 0.14                               Intestines                                                                             1.92 ± 0.26                                                                            0.56 ± 0.12                                                                            0.06 ± 0.03                               Tumor/Blood                                                                            0.07 ± 0.38                                                                            0.82 ± 0.07                                                                            0.64 ± 0.09                               Tumor/Muscle                                                                           2.7 ± 1.4                                                                              2.51 ± 0.54                                                                            1.85 ± 0.37                               n        3           3           4                                            ______________________________________                                    

Surprisingly, low molecular weight diagnostic agents when complexed tofolate and administered to animals were found to produce significantlyhigher tumor to normal tissue biodistribution ratios. Since the uptakeof folate conjugates is mediated by receptor mediated endocytoticmechanisms, and these mechanisms are generally capable of internalizinglarge macromolecules, one would not expect that lower molecular weightfolate conjugates would be more effective than high molecular weightfolate conjugates. In particular radionuclides complexed to folate via achelating agent show a high affinity for FBP receptors and thus areexcellent compounds for diagnostic imaging. In accordance with thepresent invention a radionuclide folate conjugate of the generalformula:

    V-Y·M

wherein

V=folate or folate receptor-binding analogs of folate;

Y=a chelating agent covalently bound to V; and

M=a radionuclide ion chelated with Y;

is used to image tumor cells in vivo.

In particular, gallium labeled folate complexes have been used in vivoin mice to image tumors, and these complexes demonstrate a particularlyhigh affinity for tumor cells. Localization of tumor masses as small asa few milligrams in size should be readily visible and may allow theirremoval before further metastases can occur.

To create an animal model appropriate for evaluation of FBP-receptortargeting in vivo, athymic mice were implanted subcutaneously with ca.4×10⁶ cells of the human KB line. Since normal mouse food contains ahigh concentration of folic acid (6 mg/kg chow), the animals used in thetumor-targeting studies were generally maintained on folate-free diet toregulate serum folate concentration closer to the 4-6 μg/L range ofnormal human serum. FIG. 2 shows the measured mouse serum folate levelsas a function of time following initiation of the folate-deficient diet.

To test the tumor-cell-selective uptake of metal-labeledradiopharmaceuticals in vivo, ˜180 μCi of ⁶⁷ Ga-deferoxamine-folateconjugate (See FIG. 3) was administered to two tumor-bearing athymicmice. Tumors were generated in athymic mice by subcutaneous injectinghuman KB cells into the dorsal-lateral region of the mice according toprocedures familiar to those of ordinary skill in the art. Afterestablishment of tumors in the mice, ˜180 μCi of ⁶⁷Ga-deferoxamine-folate conjugate was administered intravenously.Approximately 45 hours post-injection, gamma images were obtained andthe tissue distribution of ⁶⁷ Ga quantitated. Upon dissection, thetumors from these two animals were found to have masses of 29.6 and 8mg, while the total body mass of these animals was 19.3 and 22.6 g,respectively. Despite the small size and sub-optimal positioning ofthese tumors relative to the kidneys, the 29.6 mg tumor was readilydetected by gamma scintigraphy. At sacrifice the 29.6 mg tumor was foundto contain 3.3% of the injected dose per gram of tumor.

To better define the ability of ⁶⁷ Ga-DF-folate to target tumor cells invivo and to confirm the role of the FBP receptor in determiningconjugate tumor uptake, a series of 17 additional athymic tumor bearingmice were studied as described in Example 26. The ⁶⁷ Ga-DF-folatecomplex was delivered intravenously to the mice and the resulting tissuedistributions of the gallium-deferoxamine-folate complex are shown inTable 3. The relatively low molecular weight ⁶⁷ Ga-deferoxamine-folateconjugates have significantly higher absolute tumor uptake of theimaging agent and much better tumor to non-target tissue contrast thanthose obtained with ¹²⁵ I labeled ribonuclease-folate complexes. At 4hours post-injection, tumor uptake of the ⁶⁷ Ga-deferoxamine-folateconjugate was 5.2±1.5% of the injected dose per gram, while ¹²⁵I-ribonuclease-folate yielded only 2.7±0.5% of the injected dose pergram. The corresponding tumor/blood ratios are 409±195 for ⁶⁷Ga-deferoxamine-folate and 1.2±0.2 for ¹²⁵ I-ribonuclease-folate and thecorresponding tumor/muscle ratios are 124±47 for ⁶⁷Ga-deferoxamine-folate and 3.4±1.6 for ¹²⁵ I-ribonuclease-folate.Through the use of a gamma camera, 8 mm tumors were easily imaged invivo using the ⁶⁷ Ga-deferoxamine-folate complex.

It is anticipated that other low molecular weight radiopharmaceuticalscan be coupled to the gamma-carboxylate of folate for imaging of tumorcells. In one embodiment, a radiolabeled peptide can be complexed tofolate. The peptide moiety of the folate-peptide complex can be selectedfrom peptides/protein fragments that bind to tumor associated receptors.Peptides having an affinity for tumor associated receptors have beenpreviously described and are known to those skilled in the art. Theconjugation of such peptides to folate, either directly or indirectlythrough a linker, can impart additional tumor affinity to the imagingagent and thus further enhance the selectivity of the imaging complexfor tumor cells.

The following examples are provided to illustrate further the method ofthe present invention.

EXAMPLE 1 RAT PHEOCHROMOCYTOMA CELL UPTAKE OF BIOTIN CONJUGATED INSULIN

Rat pheochromocytoma (PC-12) cells were obtained from America TypeCulture Collection and were grown (37° C., 5% CO₂ in humidified air)attached to plastic flasks for 2 to 3 weeks until confluent in a mediumof 85% RMPI 1640, 10% v/v heat inactivated horse serum, and 5% fetalcalf serum containing 1% streptomycin-penicillin.

Biotin and fluorescein labeled insulin was prepared. To 1 ml of a 1mg/ml solution of insulin protein in phosphate buffered saline was addedsimultaneously 100 l of a 1 mg/ml solution of fluorescein isothiocyanate(FITC) in dimethylformamide (DMF) and 100 l of a 1 mg/ml solution ofN-hydroxysuccinimido biotin in dimethylsulfoxide (DMSO). The twolabeling reagents were allowed to react at room temperature for 4 hours,after which the unreacted reagents were quenched with 10 l ethanolamine.The quenched reaction mixture was then dialyzed against double distilledwater until unreacted fluorescein derivatives no longer dialyzed intothe water. The covalent attachment of biotin and fluorescein to thedesired protein was confirmed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis and western blot analysis.

As a control, non-biotinylated fluorescein labeled insulin was prepared.1 ml of a 1 mg/ml solution of insulin was added 0.5 ml of a 1 mg/mlsolution of fluorescein isothiocyanate (FITC) in dimethylformamide(DMF). The reaction was allowed to proceed for 4 hours in the dark atroom temperature. After 4 hours the reaction was quenched with 10 lethanolamine, and the labeled insulin solution was dialyzed againstdouble distilled water until unreacted FITC no longer appeared in thesolution.

The rat PC12 cells were grown in modified RMPI 1640 medium as amonolayer on the bottom of a culture flask. Before removing the cells,the monolayer was washed with a 20 ml portion of fresh Locke's solution.The cells were then displaced into 20 ml of the Locke's solution bygentle agitation with a stream Locke's solution. The suspended cellswere pelleted by centrifugation at 10,000×g for 10 seconds and afterresuspending in Locke's solution in separate polycarbonate tubes (40ml/tube) to a final density of 1.14×10⁶ cells/ml, the following amountsof proteins were added to the cell suspensions: 40 g fluorescein-labeledinsulin was added to the first tube, and to the control tube was added40 g biotin-conjugated insulin labeled with fluorescein. The tubes wereallowed to incubate at 37° C. At intervals of 5, 15 and 33 minutes, 0.5ml of each cell suspension was removed and pelleted at 10,000×g for 10seconds. The cell pellet was washed and repelleted twice in 1 ml Locke'ssolution and then fixed by addition of 200 l of a 2% formalin solutionin phosphate buffered saline. Thirteen microliters of the fixed cellsuspension was then added to a microscope slide and viewed with thefluorescent microscope to detect internalized proteins. No evidence ofinternalization was noted for the fluorescein labeled insulin acting asa control. Cellular internalization was indicated for the biotinylatedinsulin labeled with fluorescein, with the amount internalizedincreasing with time.

EXAMPLE 2 RAT PHEOCHROMOCYTOMA CELL UPTAKE OF BIOTIN CONJUGATEDHEMOGLOBIN

Following the same general procedure set forth in Example 1 hemoglobinwas biotinylated, and the biotinylated form was shown to bepreferentially internalized by rat pheochromocytoma cells as compared tonon-biotinylated hemoglobin.

EXAMPLE 3 SOYBEAN CELL UPTAKE OF BOVINE SERUM ALBUMIN

Soybean cell suspension cultures of Glycine max Merr Var Kent weremaintained by transferring cells to fresh W-38 growth medium every 7days.

To 20 ml of a suspension culture of soybean cells was added 10 g ofeither fluorescein-labeled (control) or fluorescein and biotin labeledbovine serum albumin. The cells were allowed to incubate for up to 6hours. At varying time intervals 1 ml of the cell suspension wasfiltered to remove the growth medium, washed with 50 ml fresh growthmedium, and resuspended in 20 ml of the same medium. The cell suspensionwas then viewed with a fluorescent microscope to determine whethercellular internalization of the labeled bovine serum albumin hadoccurred. Cellular internalization was indicated only for biotinylatedbovine serum albumin.

EXAMPLE 4 SOYBEAN CELL UPTAKE OF INSULIN

Following the same general procedure set forth in Example 3 insulin wasbiotinylated, and the biotinylated form of insulin was shown to bepreferentially internalized by soybean cells as compared tonon-biotinylated insulin.

EXAMPLE 5 SOYBEAN CELL UPTAKE OF HEMOGLOBIN

Following the same general procedure set forth in Example 3 hemoglobinwas biotinylated, and the biotinylated form of hemoglobin was shown tobe preferentially internalized by soybean cells as compared tonon-biotinylated hemoglobin.

EXAMPLE 6 CARROT CELL UPTAKE OF BOVINE SERUM ALBUMIN

Carrot cells of wild type origin were established and maintained in MSgrowth medium supplemented with 0.1 mg/L 2,4-dichlorophenoxyacetic acid.Bovine serum albumin was labeled with fluorescein alone as a control orwith fluorescein and biotin following the procedures detailed in Example3. The carrot cells were then incubated in the presence of therespective labeled bovine serum albumin for 7 hours. All otherconditions were the same as those described in Example 3 above. Cellularinternalization was found only in those cells contacted with biotinlabeled bovine serum albumin.

EXAMPLE 7 CARROT CELL UPTAKE OF INSULIN

Following the same general procedure set forth in Example 6 insulin wasbiotinylated, and the biotinylated form was shown to be preferentiallyinternalized by carrot cells as compared to non-biotinylated insulin.

EXAMPLE 8 CARROT CELL UPTAKE OF HEMOGLOBIN

Following the same general procedure set forth in Example 6 hemoglobinwas biotinylated, and the biotinylated form was shown to bepreferentially internalized by carrot cells as compared tonon-biotinylated hemoglobin.

EXAMPLE 9 SOYBEAN CELL DEGRADATION OF HEMOGLOBIN

To determine whether hemoglobin was rapidly degraded following cellularinternalization by transmembrane transport, soybean cells were allowedto internalize and metabolize biotinylated hemoglobin for a period of 8hours under conditions described in Example 5, after which the soybeancells were rapidly homogenized in a sodium dodecyl sulfate solution todisaggregate and denature all protein material. The solubilizedpolypeptides were separated according to molecular weight bypolyacrylamide gel electrophoresis and then electroblotted ontonitrocellulose paper. The positions of the biotin-labeled peptides werethen visualized on the nitrocellulose blot by staining with horseradishperoxidase-linked avidin and the colored substrate, p-chloronaphthol.All of the biotin-linked material was found to migrate with an apparentmolecular weight of 16,000 daltons, about equal to the molecular weightof the parent globin chains of hemoglobin, indicating no breakdown ofthe parent globin chains had occurred during the 8 hour incubationperiod.

EXAMPLE 10 IN VIVO DELIVERY TO MICE OF SOYBEAN TRYPSIN INHIBITOR

Soybean trypsin inhibitor (SBTI) (6 mg) was labeled with radioactive ¹²⁵I using 8 iodobeads (Bio Rad) in 1 mL buffer which was then dialyzed toremove unreacted ¹²⁵ I. After dividing into two equal fractions, onefraction was biotinylated with N-hydroxysuccinimidyl biotin and theother fraction was left as an unmodified control. Mice (25 g) were theninjected with either the biotinylated SBTI or the control SBTI byinsertion of a hypodermic syringe containing a 25 gauge needle into thetail vein of the mouse. After 15 minutes, each mouse was sacrificed andthen perfused with heparin-containing isotonic saline via the directcardiac influx and efflux method. When the various tissues appeared tobe blood-free, the perfusion was terminated and each tissue/organ wasremoved, weighed, and counted for ¹²⁵ I-SBTI in a gamma counter.Although some radioactivity was detected in the mice treated withnon-biotinylated ¹²⁵ I-SBTI, between 4 and 100 times more ¹²⁵ I-SBTI wasfound in the mice treated with biotinylated SBTI, indicating successfulin vivo delivery to murine cellular tissue.

    ______________________________________                                                   Counts per minute/gram wet weight                                  Tissue       Control SBTI                                                                             Biotin SBTI                                           ______________________________________                                        Liver        535        1967                                                  Lung         107        2941                                                  Kidney       5152       8697                                                  Intestine    0          700                                                   Muscle       0          1065                                                  Heart        0          739                                                   Brain        0          267                                                   ______________________________________                                    

EXAMPLE 11 SOYBEAN CELL UPTAKE OF SALMON SPERM DNA

Protein free salmon-sperm DNA, either in a highly polymerized form(≧50,000 base pair length) or in a sheared form (≦500 base pair length),was transaminated at the cytosine residues. The transaminated DNA (1 mg)was labeled with fluorescein via the addition of 0.5 mg of fluoresceinisothiocyanate (FITC) in dimethylsulfoxide (DMSO). The resultingreaction mixture was divided into two portions and the labeling reactionwas quenched in one portion by addition of 10 L of ethanolamine. Thisquenched portion served as the non-biotinylated control. The remainingDNA was then covalently labeled with biotin via reaction with 0.5 mg ofN-hydroxysuccinimidyl biotin in DMSO. After purification, the twoderivatives (1 g/ml) were separately incubated with soybean suspensionculture cells at room temperature for 6 hours and then the cells werewashed with 50 ml fresh growth medium and observed by fluorescencemicroscopy. Only the biotinylated DNA entered the soybean cells.

EXAMPLE 12 E. COLI TRANSFORMATION AND EXPRESSION OF AMPICILLIN RESISTANTGENE

Plasmid DNA (pUCS) was biotinylated via nick translation in the presenceof biotin-14-dATP using a commercially available nick translation kit(Bethesda Research Laboratories). The biotinylated DNA and unmodifiedDNA (1 g) were added to E. coli strain Cu 1230 that had been madecompetent by treatment with MgCl₂ and CaCl₂ following the method ofManiatis et al., Molecular Cloning: A Laboratory Manual, pp. 250-251,Cold Spring Harbor Press (1987). After transformation, the successfultransformants were selected by plating cells on LB media which contained50 g/ml ampicillin and then incubated overnight at 37° C. Colonies whichsurvived the ampicillin were counted and the transformation efficiencywas determined. The number of surviving E. coli colonies was at least100-fold greater in E. coli transformed with the biotinylated plasmids.

EXAMPLE 13 BLOCKADE OF DELIVERY OF BIOTINYLATED PROTEINS INTO SOYBEANCELLS BY COMPETITION WITH UNLIGATED BIOTIN

Insulin, ribonuclease (RNase) and bovine serum albumin (BAS) wereindividually biotinylated following the same general procedure set forthin Example 1 above. A sample of each of the biotinylated proteins and anunmodified sample of the same protein (control protein) wereradioiodinated according to the following protocol. To 1 mL of a 200 mMphosphate buffer, pH 7.0, containing 3 iodobeads (Pierce Chemical Co.)was added 0.2 mCi ¹²⁵ I!-NaI (carrier-free in 1 n NaOH, Amersham) andthe mixture was allowed to incubate for 5 minutes to liberate the activeiodine species, according to the supplier's instructions. Afteractivation, 1 mg of desired biotinylated or control protein was added in0.5 mL of iodination buffer. The iodination was allowed to proceed withstirring for 20 minutes. After the iodination was complete, the productwas isolated via gel filtration on a Biogel PH-10 column. Typicaliodinations of ribonuclease A (Sigma Chemical Co.) yielded a productemitting 2×10⁵ cpm/g.

Uptake of ¹²⁵ I-labeled proteins by soybean suspension culture cells inthe early exponential growth phase was then assayed as follows. To eachculture was added sufficient ¹²⁵ I-labeled macromolecule to achieve afinal concentration of 10 g/mL, and the suspension was incubated at 23°for the desired time. After the desired incubation period, the cellswere washed for 5 minutes in growth media rebuffered to pH 8 with 15 mMglycylglycine to remove surface bound ligand. The cell suspension wasthen filtered, washed with 200 volumes growth media, and placed incounting vials.

Uptake of biotin-conjugated RNase was rapid, reaching 6×10⁶ moleculesinternalized per cell in the first 3 hours. In contrast, unmodifiedRNase was not internalized, demonstrating the importance of the biotinadduct. To further confirm the role of biotin in mediating the deliveryof RNase, the cell suspension was treated with 1 mM free biotin directlyprior to addition of the biotin-derivatized RNase. Free biotincompetitively blocked delivery of the conjugated protein into thesoybean cells. Therefore, it can be concluded that the internalizationprocess involves recognition of biotin by a limited number of receptorson the plant cell surface.

Similar studies with biotin-labeled BSA and insulin yielded virtuallyidentical results.

EXAMPLE 14 PARTIAL PURIFICATION OF BOVINE SERUM ALBUMIN FOLLOWING ITSINTERNALIZATION BY CULTURED SOYBEAN CELLS

Radiolabeled, biotinylated bovine serum albumin was allowed to bind andenter cultured soybean cells following the same general procedure setforth in Example 13, after which the cells were thoroughly washed,homogenized and extracted to remove cytoplasmic soluble proteins. Thiscytoplasmic protein extract was separated using standard chromatographictechniques on a Sephadex G-25 gel filtration column to determine whetherany small molecular weight fragments might be generated during theco-delivery process. Comparison of the elution profile of the-¹²⁵I-labeled material isolated from the cell extract with the profile ofunmodified ¹²⁵ I-serum albumin showed that the majority of theinternalized protein remained intact throughout the 2 hour duration ofthe internalization study.

EXAMPLE 15 RESTORATION OF GROWTH IN CULTURED CELLS DEFICIENT INHYPOXANTHINE-GUANINE PHOSPHORIBOSYL TRANSFERASE (HGPRT) UPON ADDITION OFBIOTINYLATED--HGPRT

Cells deficient in HGPRT (i.e., the defect in Lesch-Nyhan Syndrome) areable to grow only in a cellular growth medium containing hypoxanthine,aminopterin and thymidine, (HAT), supplemented with purines. However,these same cells were found to grow normally in HAT medium afterinternalization of biotin-linked HGPRT via the biotin-mediatedendocytosis pathway. HGPRT was biotinylated in the presence ofhypoxanthine and phosphoribosyl pyrophosphate (to protect the activesite) with N-hydroxysuccinimido biotin. The crosslinked enzyme retained55% of the original activity and SDS PAGE analysis followed bytransblotting and avidin-peroxidase binding indicated that a 1-4 biotinswere attached per molecules of HGPRT. HGPRT deficient fibroblasts (GM00152) incubated with biotinylated HGPRT (4.6×10⁴ units/cell) grew at arate comparable to cells supplemented with purines for at least 24hours. Appropriate control incubations did not grow on HAT mediumsupplemented with HGPRT, biotin, phosphoribosyl, and inosinemonophosphate.

EXAMPLE 16 TRANSFORMATION OF CULTURED SOYBEAN CELLS WITH A KANAMYCINRESISTANCE GENE USING THE BIOTIN DELIVERY SYSTEM

The expression vector pGA642-643 containing a bacterial kanamycinresistance gene was nicked with EcoR1 and the sticky ends were filled inusing biotinylated ATP and a T4 polymerase-based nick translation kitfollowing the general procedure set forth in Example 12. Identicalcontrol plasmids were left unmodified. Then, to 40 ml of a soybean cellsuspension was added either the biotinylated plasmid or the control(nonbiotinylated) plasmid. After incubation for 10 hours, the cells fromeach flask were transferred to fresh growth medium containing 100 g/mlkanamycin and allowed to proliferate under normal conditions. Each flaskwas also transferred to fresh medium containing 100 g/ml kanamycin every3 days. By day 10, the flask treated with the biotinylated plasmid hadincreased 6-fold in cell mass, while the flask treated with the controlplasmid exhibited no measurable growth.

EXAMPLE 17 USE OF FOLIC ACID CONJUGATION TO DELIVER RIBONUCLEASE INTOCULTURED HUMAN CELLS

Activated folic acid was prepared by dissolving 1 mg of folic acid and3.8 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)in 0.5 ml of dimethylsulfoxide (DMSO). The solution was allowed to setfor 2.5 hours. A sample of folate-labeled bovine ribonuclease wasprepared by treating the ribonuclease with 34-fold molar excess ofEDC-activated folate. The resulting derivatized RNase contained 12-14covalently bound folates per protein molecule. A second sample of theribonuclease was left unmodified to serve as a control. Thefolate-labeled sample and the control sample were radioiodinatedfollowing the same general procedure set forth in Example 13. Followingexhaustive dialysis, the two ¹²⁵ I-labeled samples were added to KBcells (a human nasopharyngeal cell line) and examined for uptake of ¹²⁵I-RNase after 30 minutes. No protein uptake was seen for RNase controlsamples, while 10⁷ molecules per cell were internalized by the RNaselabeled with folate (RNase-Folate). To confirm that the uptake wasindeed folate-mediated, the KB cells were treated with either controlRNase or folate-labeled RNase in the presence of a 100-fold molar excessof unligated folate (100×). The control RNase again displayed nointernalization; uptake of the RNase-Folate conjugate was reduced 7-foldby competitive inhibition. Similar studies yielded corresponding resultsusing human HeLa cells.

EXAMPLE 18 USE OF FOLIC ACID CONJUGATION TO DELIVER SOYBEAN TRYPSININHIBITOR (SBTI) INTO CULTURED HUMAN CELLS

Experiments following the general procedure set forth in Example 17,with soybean trypsin inhibitor being substituted for ribonuclease, wereconducted with virtually identical results. Folate ligation was againdemonstrated to be essential for uptake of SBTI by KB cells.

EXAMPLE 19 VISUALIZATION OF RIBONUCLEASE ENDOCYTOSIS BY KB CELLS USING ACONFOCAL MICROSCOPE

Bovine ribonuclease (RNase) was labeled with fluorescein isothiocyanatefollowing the same general procedure set forth in Example 1 and thenfurther labeled with folate following the same general procedure setforth in Example 17. RNase labeled only with fluoroscein was used as acontrol. Following extensive dialysis against growth medium, the controland folate-labeled RNase samples were added to separate cultures of KBcells. After 60 minute incubation, the cells were thoroughly washed andexamined for uptake. Only the folate-labeled samples displayed anyinternal fluorescence when viewed with laser excitation under theconfocal microscope (Bio Rad). Furthermore, using the confocal'scapability of focusing on a single horizontal plane in each culturedcell, it was readily evident that vesicles filled with thefluorescent-labeled, folate-bound ribonuclease were forming on allregions of the cell surface, pinching off via endocytosis into theinterior, and entering the cytoplasm. The vesicles, measuring 0.8 to 1.0m across, were easily large enough to accommodate large biomoleculessuch as proteins and DNA plasmids.

EXAMPLE 20 UPTAKE OF RIBONUCLEASE IN COMPLEX WITH FOLATE BY WHITE BLOODCELLS

Fluorescein-labeled RNase was either conjugated to folate or leftunmodified (control) following the same general procedure set forth inExample 19. The folate-conjugated and control samples were then added tofreshly drawn whole human blood, incubated at 37° C. for 2 hours andthen washed thoroughly and examined under the fluorescence microscope.Cells bearing folate receptors that were brought into contact with theRNase/folate/fluorescein complex were found to fluoresce. None of thecontrol cells exhibited fluorescence.

EXAMPLE 21 IN VIVO DELIVERY OF RIBONUCLEASE THROUGHOUT TISSUES OF LIVEMICE FOLLOWING INTRAVENOUS INJECTION

Ribonuclease was labeled with ¹²⁵ I following the same general procedureset forth in Example 13 and then further conjugated with folate or leftunmodified to serve as a control, following the general procedure setforth in Example 17. Live mice were injected with either thefolate-conjugated or control sample by inserting a 27 gauge needle intothe tail vein of the mice and injecting 0.2 ml of the appropriate sampledissolved in physiological saline. After 1 hour, the mice wereanesthetized, perfused with saline and dissected to determine thespecific radioactivity of each organ, following the general procedureset forth in Example 10. Uptake was determined by relative comparison ofspecific radioactivity of the various tissues examined (units comparedwere counts per minute/gram of tissue divided by the specific activityof a blood sample drawn 3 minutes after injection, i.e., in order tonormalize for any variability in the amount injected). Folateconjugation provided greatly enhanced uptake by the liver and lung,while the kidney, an organ responsible for clearance of unwantedproteins, was enriched in unmodified RNase.

Similar results were obtained when the mice were allowed to live for 18hours post-injection, with preferential uptake of folate-conjugatedRNase also being noted in the intestine, heart, muscle and brain.

EXAMPLE 22 IN VIVO DELIVERY OF RIBONUCLEASE THROUGHOUT TISSUES OF LIVEMICE FOLLOWING INTRAPERITONEAL INJECTION

Folate-derivatized and control RNase (¹²⁵ I-labeled) were prepared asdescribed in Example 21 and injected into the peritoneal cavity of 30 gmice using a 27 gauge needle and syringe. After 17 hours, the mice wereanesthetized, perfused, and dissected to remove various body tissues.Each tissue was then weighted and counted for radioactivity. Thespecific uptake of both the control and folate-conjugated RNase werecompared following the general procedure set forth in Example 21. Ascompared to intravenous administration, intraperitoneal injectionresulted in enhanced delivery of the folate-derivatized RNase to alltissues except the kidney. Transmembrane delivery across the blood/brainbarrier was demonstrated by the brain tissue's preferential uptake ofthe folate-labeled protein. Similar results were obtained in two otherrepetitions of the foregoing procedure.

EXAMPLE 23 REVERSION OF src-TRANSFORMED FIBROBLASTS TO DIFFERENTIATEDSTATE UPON TREATMENT WITH ANTI-SENSE DNA CONJUGATED TO FOLATE

A pentadecameric oligonucleotide DNA probe of the formula ##STR1##complementary to a sequence spanning the initiation codon of the Roussarcoma src oncogene and containing a free 3' amino group wasderivatized with folate using carbodiimide chemistry. A second samplewas left unmodified as a control. Both samples were dissolved inphosphate buffered saline and introduced into culture dishes containingfibroblasts transformed by the Rous sarcoma virus (XC cells) at a finaloligonucleotide concentration of 8×10⁻⁶ M. After 24 hours, the culturedcells were viewed under a microscope. Results showed that 40% of thecells treated with the folate/antisense oligonucleotide complex hadreverted to the normal fibroblast-like morphology, while only 10% of thecontrols displayed the same nontransformed phenotype. The remainingcells in both culture dishes retained their highly rounded shapecharacteristic of the neoplastic state.

EXAMPLE 24 RIBOFLAVIN ENHANCED UPTAKE OF MACROMOLECULES

Methods of conjugating riboflavin to macromolecules

Three methods have been used to covalently link riboflavin to aproteins. All 3 methods have been shown to enable the delivery ofattached macromolecules as diverse as BSA (Mr-68,000), momordin(Mr-22,000) and ribonuclease (Mr-13,700) nondestructively into livingcells (vide infra). The first method involves oxidation of the sidechain of riboflavin first with periodate and then further withpermanganate, followed by carbodiimide mediated coupling of thegenerated carboxylate to N-hydroxysuccinimide (NHS). The NHS ester ofthe modified riboflavin can then react with any primary or secondaryamine, such as the lysine side chains present on the surface of BSA.

The NHS-riboflavin can be reacted with cysteamin to generate ariboflavin derivative with a free sulfhydryl at the end of a shortspacer. This spacer is then lengthened by reaction of the sulfhydrylwith maleinidylbenzoyl NHS. The resulting NHS derivative of riboflavinis similarly reactive toward primary amines, only the vitamin isseparated from the conjugated protein by a 12 atom spacer.

A different reaction scheme can be used to conjugated riboflavin to aprotein or other macromolecule as follows. Unmodified riboflavin isfirst reacted with succinic anhydride to extend the vitamin at the siteof the primary hydroxyl. The free carboxylate is then activated asdescribed above with NHS in the presence of a carbodiimide. Theresulting derivative can link riboflavin to any amine-containingmolecule by a 5 atom spacer.

Quantitative analysis of ribonuclease-riboflavin uptake by BHK cells

Ribonuclease was labeled with ¹²⁵ I as described by Leamon and Low(1991) Proc. Natl. Sci. USA 88, 5572-5576, and then either furtherderivatized with riboflavin or left unmodified. The samples (10 μg/ml)were then added to 50% confluent monolayers of BHK cells and incubatedfor various times. At various times as shown on the abscissa of FIG. 4,the cells were washed 5× in saline, and removed and counted in a gammacounter. The ribonuclease sample derivatized with 6 riboflavins perprotein (RNase A-Rf (6)) resulted in the highest rate of uptake followedby the sample conjugated to just one riboflavin (RNase A-Rf (1)). Thetwo samples lacking riboflavin (RNase-control (6) and RNase-control (1))endocytosed little or no ¹²⁵ I-ribonuclease. (see FIG. 4)

Analysis of riboflavin-mediated protein uptake by various tissues inlive rats

¹²⁵ I-BSA(40 μg) derivatized further with riboflavin (2.3 moles/moleBSA) or left unmodified was injected into the tail vein of Wistar whitefemale rats and allowed to circulate 1 hr. The rats were thenanesthetized, perfused through the left ventricle of the heart withphosphate buffered saline containing 10 U/ml heparin until the effluentfrom the right ventricle appeared clear and the organs appeared pale,and the rates were then dissected. The specific radioactivities of eachorgan were then determined see FIG. 5, the solid bars represent the BSAcontent of tissues from rats treated with the ¹²⁵ I-BSA-riboflavinconjugated samples, while the open bars represent the controls (¹²⁵I-BSA)!. Clearly, conjugation with riboflavin enhances tissueretention/uptake manyfold in live rats.

EXAMPLE 25 THIAMIN ENHANCED UPTAKE OF MACROMOLECULES

Protocol for Thiamin Coupling

Thiamin was dried at 95° C. for 4 hours. To 200 mg dry thiamin 320 μlthionyl chloride was added with stirring in an ice bath for 10 minutesand then 40 μl pyridine was added in same way. The mixture was stirredat 0° C. for 10 minutes and then was moved to a 50° C. oil bath andreacted for an additional 60 minutes. White thiamin was washed withether 4 times and then dried by aspiration for a half hour. The dryproduct was stored at 4° C.

10 mg of activated thiamin in small aliquots was added to 1 ml of a 10mg/ml Bovine Serum Albumin/PBS solution. The pH of the mixture wasmaintained at 8. After stirring for 10 minutes the solution changed todeep green color. The mixture was centrifuged at 5000 g. for 20 minutesat 4° C. The liquid phase containing the labeled BSA was saved forpurification.

The labeled BSA was purified by G-75-120 gel filtration chromatographyin pH 7.4 PBS two times. The fast moving green band was the BSA-Thiamin.The amount of thiamin attached to BSA was estimated by oxidizing theconjugated thiamin to thiochrome and measuring the level of fluorescenceat excitation 365 nm and emission 445 nm. The oxidation of theconjugated thiamin was preformed as follows: 20 μl BSA-thiamin solutionwas diluted to 2 ml with dH₂ O. 20 μl 2% K₃ Fe (CN) 6 and 20 μl 1M NaOHwere added to the above solution. The mixture was vortexed for 10seconds, allowed to stand for 10 minutes, then evaluated for the levelof fluorescence. BSA concentration was estimated by the BicinchoninicAcid (BCA) method.

0.5 mg fluorescein isothiocyanate/100 μl dimethyl formamide was added to1 ml of a 2 mg BSA-thiamin/ml solution. The pH of the mixture wasmaintained at 8 while the reaction was stirred at room temperature for 3hours. FITC-BSA-thiamin was separated from free FITC by two rounds ofG-75-120 gel filtration chromatography. The average number of FITCmolecules conjugated to BSA was estimated by measuring the absorbance at495 nm.

Cell Culture and Uptake Protocol

KB Cells were cultured in thiamin deficient Eagle's minimum essentialmedium consisting of 10% fetal bovine serum (heat denatured), 100Units/ml penicillin, 100 μg/ml streptomycin, 2 μg/ml amphotericin B and2 mM glutamine for more than two passages. KB Cells were transferred to6 cm culture dishes and cultured at 37° C. for two days (until about 90%confluent). The medium in each dish was replaced with 2 ml new thiamindeficient MEM and the cells were incubated with BSA-thiamin, as desired.

Treated cells were cultured at 37° C. for 3 hours. The culture disheswere then washed with PBS four times and the cells of each dish werescraped and collected into a 1 ml centrifuge tube. The cells in thetubes were washed with PBS 4 times, and then spun down and lysed with 1ml 1% Triton-X 100.

The lysis solutions were assayed for fluorescence at excitation 495 nmand emission 520 nm, and the protein content was estimated by theBicinchoninic Acid (BCA) method. From the fluorescence one can calculatethe total number of BSA molecules taken up by the KB cells in each dish,and from the protein concentration one can calculate the cell number ineach dish. The ratio of the two measurements yields the number ofBSA-thiamin molecules internalized per cell.

EXAMPLE 26 THIAMIN & RIBOFLAVIN UPTAKE BY A549 CELLS

Observation of thiamin & riboflavin mediated protein uptake byfluorescence microscopy

Bovine serum albumin was labeled, with fluorescein isothiocyanate (FITC)and optionally further complexed with either thiamin or riboflavin. A549cells were incubated with FITC-BSA, FITC-BSA-thiamin orFITC-BSA-riboflavin in accordance with the procedure described above.The accumulated data is represented by FIG. 6. As the data indicates,the uptake of BSA was enhanced when BSA is conjugated with thiamin orriboflavin as compared to BSA conjugated to FITC alone. Applicants havealso discovered that serum contains a binding protein that competes withthe cellular receptors for thiamin. Removal of serum prior to incubatingthe cells with thiamin conjugated BSA, further enhances the uptake ofthe conjugated BSA complex.

Time Dependent of Uptake of BSA and BSA-Thiamin by KB Cells

The time-dependent uptake of thiamin-BSA-FITC conjugates by KB cells hasbeen measured. (See FIG. 7). Solid circles represent the FITC-BSAconjugate, and the solid squares and solid triangles representthiamin-BSA-FITC conjugates, wherein the solid squares represent anaverage of 1.8 molecules of thiamin per BSA molecule and the solidtriangles represent 3.9 molecules of thiamin per BSA molecule. As thedata indicates both thiamin-BSA-FITC conjugates are taken up to a muchgreater extent than the BSA-FITC conjugates.

EXAMPLE 27 PREPARATION AND PURIFICATION OF FOLATE-DEFEROXAMINECONJUGATES

Materials. Folic acid, deferoxamine (DF) mesylate, and DEAE-trisacrylanion-exchange resin were purchased from Sigma (St. Louis, Mo.).Bicinchoninic acid (BCA) protein assay kit was obtained from Pierce(Rockford, Ill.). Acetonitrile (HPLC grade) and dicyclohexylcarbodiimide(DCC) were purchased from Aldrich (Milwaukee, Wis.). Gallium-67 chloridewas purchased from Mallinckrodt Medical, Inc. (St. Louis, Mo.). Tissueculture products were obtained from GIBCO (Grand Island, N.Y.), andcultured cells were received as a gift from the Purdue Cancer Center(West Lafayette, Ind.).

100 mg DF mesylate was dissolved in 3 mL dimethylsulfoxide containing200 μL pyridine. A 10-fold excess of folic acid (672 mg) was dissolvedin 15 mL warm (˜40° C.) dimethylsulfoxide and 5 molar equivalents of DCC(157 mg) were then added. The reaction mixture was stirred at 40° C. inthe dark, during which ninhydrin assay and thin layer chromatographywere used to follow the reaction process. After the coupling wascomplete, the DF-folate conjugate and excess folic acid wereprecipitated with 200 mL cold acetone and pelleted by centrifugation.The pellet was washed once with cold acetone, dried under vacuum andthen redissolved in 5 mL deionized water. The pH of the solution wasadjusted to 8.0 to facilitate dissolution of the solid.

The crude product contained a mixture of folate-deferoxamine conjugates,wherein the folate is linked to DF via its α-carboxyl or γ-carboxylgroup as well as unreacted folic acid. The two isomers of DF-folate wereisolated and purified on a weak anion-exchange column. Briefly, theproduct mixture was loaded onto a 1.5 cm×15 cm DEAE-trisacryl columnpre-equilibrated in 10 mM NH₄ CO₃ buffer (pH 7.9). The column was washedwith 50 mL 10 mM NH₄ HCO₃ and then eluted with a 500 mL gradient of80-180 mM NH₄ HCO₃ followed by 150 mL 500 mM NH₄ HCO₃. Threefolate-containing peaks were obtained as detected by UV absorbance at363 nm. Each peak was collected, lyophilized, and redissolved indeionized water. The purity of each component was confirmed byreverse-phase high pressure liquid chromatography (HPLC) with a 10mm×250 mm Licrosorb RP-18 column (Altech, Deerfield, Ill.), andevaluation of the conjugates' molecular weights was determined byfast-atom bombardment mass spectroscopy (FAB-MS). The characteristic pKavalues of the two DF-folate isomers were obtained by titration on apH/ion analyzer (Corning, Corning, N.Y.).

The first two peaks were identified as DF-folate conjugates, both givinga molecular weight of 984.0 in their FAB-MS spectra indicating theexpected 1:1 ratio between folic acid and DF. The third peak showed thesame molecular weight as free folic acid. Since the two isoforms ofDF-folate conjugate retain either a free γ-carboxyl or free α-carboxyl,they can be distinguished from each other and from unreacted folic acidby their characteristic pKa values, which were determined by titration.The DF-folate(α) conjugate (pKa=2.5, constituting ˜20% of totalDF-folate) eluted in 140-260 mL fractions (peak 1), the DF-folate(γ)conjugate (pKa=4.5, constituting ˜80% of total DF-folate) eluted in 340mL to 420 mL fractions (peak 2), and the free folic acid (pK_(a1) =2.5,pK_(a2) =4.5) eluted between 580 mL to 680 mL (peak 3).

EXAMPLE 28 PREPARATION OF ⁶⁷ GA-RADIOTRACERS

⁶⁷ Ga-deferoxamine-folate conjugate, ⁶⁷ Ga-citrate, and ⁶⁷Ga-deferoxamine were prepared from no-carrier-added ⁶⁷ Ga-gallium(III)chloride (Mallinckrodt Medical, Inc. St. Louis, Mo.). The ⁶⁷Ga-deferoxamine-folate conjugate was prepared as follows: A dilute HClsolution of ⁶⁷ Ga³⁺ was evaporated to dryness with heating under astream of N₂ and the tracer reconstituted in 300 μL ethanol containing0.002% acetylacetone (acac). The ethanolic ⁶⁷ Ga(acac)₃ solution (3.2mCi) was diluted with an equal volume of TRIS-buffered saline (pH 7.4)followed by addition of 2.25×10⁻⁶ mole of aqueous DF-folate(γ)conjugate. Labeling was complete after standing at room temperature for18-24 hours.

⁶⁷ Ga(III)-citrate was prepared by evaporating a ⁶⁷ Ga-chloride solutionto dryness and reconstituting with 0.1 mL of 3% sodium citrate (pH 7.4).A portion of the resulting ⁶⁷ Ga-citrate solution (50 μL) was mixed with0.1 mg deferoxamine to obtain ⁶⁷ Ga-deferoxamine (⁶⁷ Ga-DF).

The radiochemical purity of the ⁶⁷ Ga-tracers was determined by thinlayer chromatography on C₁₈ reverse phase silica gel plates eluted withmethanol and in all cases was found to exceed 98%. Theradiochromatograms were evaluated using a Berthold (Wildbad, Germany)Tracemaster 20 Automatic TLC Linear Analyzer. Rf values of 0.93; 0.0;0.1; and 0.74 were obtained for ⁶⁷ Ga-DF-folate(γ); ⁶⁷ Ga(acac)₃ ; ⁶⁷Ga-DF; and ⁶⁷ Ga-citrate, respectfully. All experiments employing the ⁶⁷Ga-DF-folate(γ) tracer were performed within 1-3 days of preparation.

EXAMPLE 29 DETERMINATION OF THE AFFINITIES OF THE TWO DF-FOLATE ISOMERSFOR CELL SURFACE FOLATE RECEPTORS

Cell Culture. KB cells, a human nasopharyngeal epidermal carcinoma cellline that greatly overexpresses the folate binding protein, werecultured continuously as a monolayer at 37° C. in a humidifiedatmosphere containing 5% CO₂ in folate-deficient modified Eagle's medium(FDMEM) (a folate-free modified Eagle's medium supplemented with 10%(v/v) heat-inactivated fetal calf serum as the only source of folate)containing penicillin (50 units/mL), streptomycin (50 μg/mL), and 2 mML-glutamine. The final folate concentration in the complete FDMEM is thephysiological range (˜2 nM). 48 h prior to each experiment, the cellswere transferred to 35 mm culture dishes at 5×10⁵ cells per dish andgrown to ˜80% confluent.

The affinity of the DF-folate(α) and DF-folate(γ) conjugates for the KBcell folate-binding protein was evaluated in a competitive binding assayusing ³ H!folic acid as the receptor ligand. Briefly, 100 pmoles of ³H!folic acid and 100 pmoles of either DF-folate(α) or DF-folate(γ)dissolved in phosphate-buffered saline (PBS) were added to KB cellsgrown ˜80% confluence in 1 mL FDMEM in 35 mm culture dishes. Following a30 min incubation at 4° C., the cells were washed 3 times with cold PBS.Cell-associated ³ H! folic acid was then determined by liquidscintillation counting, and the cellular protein content was evaluatedby the BCA protein assay.

Assuming a cellular protein content of ˜2×10⁻⁷ g, 4.85×10⁻⁶ folatereceptors were occupied with the radiolabeled ligands on each cell. A50% decrease in bound ³ H!folic acid was observed in the presence of anequimolar amount of the DF-folate(γ) conjugate, while the DF-folate(α)conjugate display no ability to compete with the radiolabeled vitamin.The competition by DF-folate(γ) was similar to that of unlabeled folicacid, indicating that covalent conjugation of DF to the γ-carboxyl offolic acid does not compromise the latter's high affinity for themembrane-associated folate binding protein.

EXAMPLE 30 UPTAKE OF ⁶⁷ GA-DF-FOLATE COMPLEX BY CULTURED KB CELLS

Because folate and its conjugates bind to cell surface receptors at 4°C., but are capable of endocytosis only at higher temperatures, it ispossible to separately evaluate the kinetics of binding theinternalization of folate by measuring the rates of folate conjugateuptake at the two temperatures. Half maximal binding of ⁶⁷Ga-DF-folate(γ) was achieved in ˜3 min at 4° C., suggesting rapidassociation of the conjugate with unoccupied receptors. By the end ofthe 30 min incubation, binding approached to saturation with ˜18% of theinitial radioactivity found associated with a cell surface.

Incubation at 37° C., a temperature which permits both binding andendocytosis, yielded similar kinetic results, however, maximal uptakereached 32% of the total conjugate added. Presumably, the difference inmagnitude of the two cellular uptake curves reflects the ability of thefolate receptor to internalize the conjugate and then recycle to thecell surface in its unoccupied form. As controls, ⁶⁷ Ga-DF lacking thefolate group did not show any significant uptake by the KB cells, nordid the ⁶⁷ Ga-DF-folate(α) complex. This latter result is consistentwith the inability of the α-conjugate to compete with free folate forthe cell surface receptor. When ⁶⁷ Ga-citrate was added to the culturemedium and incubated at 37° C. for 30 min, cell-associated ⁶⁷ Garadioactivity was 106-fold lower than observed with ⁶⁷ Ga-DF-folate(γ).

To verify the involvement of a cell surface folate receptor in mediatingthe uptake of ⁶⁷ Ga-DF-folate(γ), binding and internalization of thecomplex were evaluated as a function of the complex's concentration.Cellular uptake of ⁶⁷ Ga-DF-folate(γ) was concentration dependent atboth 4° C. and 37° C., saturating at levels of 20% and 35% of the totalradioactivity added, respectively. Analysis of competition between ⁶⁷Ga-DF-folate(γ) and unlabeled folic acid further demonstrated thatcellular uptake was folate receptor-mediated by specific binding, sinceonly 0.5% of initial cellular uptake was retained in the presence of100-fold molar excess of free folate. A 50% decrease in uptake was againobserved when equimolar amounts of ⁶⁷ Ga-DF-folate(γ)/DF-folate(γ) andunlabeled folic acid were mixed and then added to the cell culture,suggesting that the affinity of the radiolabeled conjugate for themembrane-associated folate receptor is comparable to that of themetal-free conjugate. In aggregate, these results suggest that ⁶⁷Ga-DF-folate(γ) associates with cell folate receptors in much the samemanner as free folic acid.

EXAMPLE 31 IMAGING WITH A ⁶⁷ GA-DEFEROXAMINE-FOLATE CONJUGATE

To test the viability of the FBP-receptor as a pathway for achievingtumor-cell-selective uptake of metal-labeled radiopharmaceuticals invivo, ˜180 μCi of ⁶⁷ Ga-deferoxamine-folate conjugate was administeredintravenously to two tumor-bearing athymic mice via the femoral veinnine days after subcutaneous injection of 4×10⁶ human KB cells. Atapproximately 45 hours post-injection, gamma images were obtained, theanimals were sacrificed and dissected, and the tissue distribution oftracer was quantified by gamma counting (after storage of the weightedtissue samples for decay to a suitable ⁶⁷ Ga count rate). The tumorsfrom the two animals were found to have masses of 29.6 and 8 mg, whilethe total body mass of the two animals was 19.3 and 22.6 grams,respectively. Despite the small size and sub-optimal positioning ofthese tumors relative to the kidneys, the 29.6 mg tumor was readilydetected by gamma scintigraphy. At sacrifice the 29.6 mg tumor was foundto contain 3.3% of the injected dose per gram of tumor, while the 8 mgtumor contained 2.8% of the injected dose per gram of tumor. The tumorto blood ratio was 1500 and 1185 and the tumor to muscle ratio was 170and 288 for each respective mouse.

EXAMPLE 32 IMAGING AND RADIOTRACER BIODISTRIBUTION STUDIES WITH A ⁶⁷GA-DEFEROXAMINE-FOLATE CONJUGATE

To better define the ability of ⁶⁷ Ga-deferoxamine-folate to targettumor cells in vivo and to confirm the role of the FBP receptor indetermining conjugate tumor uptake, an additional study was conductedusing 17 athymic mice. Male athymic mice (Nu/Nu; 21-28 days old) werehoused under aseptic conditions, and fed a folate-deficient diet fromthe time of receipt, unless otherwise indicated. Folate-deficient rodentchow was obtained from ICN Biochemicals and autoclaved prior to use.Animals were anesthetized with ketamine (40 mg/kp, i.p.) and xylazine (4mg/kg, i.p.) for radiopharmaceutical injection, for gamma imagingstudies, and again prior to sacrifice. Syringes used for radiotracerinjections were weighted on an analytical balance before and afterinjection to quantitate the dose received by each animal.

A Capintec CRC12R Radionuclide Dose Calibrator was used for appropriateassays of ⁶⁷ Ga; while samples requiring precise quantitation of ⁶⁷ Gawere counted in a Packard 5500 Automatic Gamma Scintillation Counterwith 3-inch large-bore NAI(Tl) crystal. Gamma images of intact animalswere obtained using a Searle 37GP gamma scintillation camera fitted witha 300 keV parallel hole collimator and linked to a Siemens MicroDELTAcomputer.

Fifteen days after subcutaneous injection of 4×10⁶ human KB cells intothe shoulder of the mice, each animal received 125-150 μCi of either ⁶⁷Ga-DF-folate (11 animals; Groups 1-4), ⁶⁷ Ga-DF (3 animals; Group 5), or⁶⁷ Ga-citrate (3 animals; Group 6) via intravenous injection into thefemoral vein. Injection volumes were approximately 130 μL of 10% ethanolin saline per animal. All animals except two were maintained onfolate-deficient diet for 3 weeks prior to radiotracer administration;the remaining two animals were maintained on normal rodent chow andincluded in the animals that received ⁶⁷ Ga-DF-folate (Group 2). Tocompetitively block tumor folate receptors three animals received2.4±1.0 mg folate intravenously ˜5 minutes prior to ⁶⁷ Ga-DF-folateadministration (Group 3). Three different animals that received ⁶⁷Ga-DF-folate also received 3.5±0.9 mg of folate intravenouslyapproximately 1 hour before being sacrificed (Group 4). The tissuedistribution of the tracers was periodically monitored by gammascintigraphy to qualitatively assess tumor uptake of tracer andtumor-background contrast. Tumor uptake was evident at one hourpost-injection, and by 3-4 hours post-injection the tracer initiallypresent in the liver had substantially cleared into the intestines. At4-4.5 hours following administration of the ⁶⁷ Ga-radiopharmaceuticalsthe anesthetized animals were sacrificed by decapitation and the tumorand major organs removed, weighed, and stored until ⁶⁷ Ga had decayed tolevels suitable for counting. The biodbiodistribution of tracer in eachsample was calculated as both a percentage of the injected dose perorgan and as a percentage of the injected dose per gram of tissue (wetweight), using counts from a weighed and approximately diluted sample ofthe original injected for reference.

A summary of the biodistribution data for the ⁶⁷ Ga-labeleddeferoxamine-folate conjugate plus the ⁶⁷ Ga-DF and ⁶⁷ Ga-citratereference tracers is presented in Tables 3 and 4. The data is alsopresented in bar graph form in FIGS. 8 and 9. FIG. 8 illustrates thepercent injected dose ⁶⁷ Ga-radiotracer per gram tumor. FIG. 9illustrates the ratio of ⁶⁷ Ga-radiotracer concentration in tumor tissuecompared to blood (% of injected dose per gram wet weight) at 4 to 4.5hours post-injection. For both FIG. 8 and 9, each bar represents thedata from one animal. Group 1 was administered ⁶⁷Ga-deferoxamine-folate; Group 2 was administered ⁶⁷Ga-deferoxamine-folate to mice maintained on a high folate diet; Group 3was administered folic acid (approximately 2.4 mg) prior toadministration of ⁶⁷ Ga-deferoxamine-folate; Group 4 was administered ⁶⁷Ga-deferoxamine-folate with a chase dose of folate one hour prior tosacrifice; Group 5 was administered ⁶⁷ Ga-deferoxamine; Group 6 wasadministered ⁶⁷ Ga-citrate.

                                      TABLE 3                                     __________________________________________________________________________    Biodistribution of .sup.67 Ga-Radiotracers in Athymic Mice with               Subcutaneous KB Cell Tumors                                                            Percentage of Injected Dose per Gram of Tissue 4 Hours Following              Intravenous Administration*                                                   .sup.67 Ga-DF-Folate.sup.†                                                                            .sup.67 Ga-DF                                                                         .sup.67 GA-citrate                     Group 1 Group 2 Group 3                                                                              Group 4.sup.‡                                                              Group 5 Group 6                       __________________________________________________________________________    Blood     0.14 ± 0.004                                                                      0.019 ± 0.011                                                                      0.45 ± 0.16                                                                       0.046 ± 0.009                                                                      0.026 ± 0.009                                                                      13.5 ± 3.2                 Heart    0.029 ± 0.004                                                                      0.024 ± 0.014                                                                      0.17 ± 0.05                                                                       0.025 ± 0.005                                                                      0.021 ± 0.002                                                                      3.4 ± 0.2                  Lungs    0.038 ± 0.005                                                                      0.063 ± 0.001                                                                      0.38 ± 0.14                                                                       0.052 ± 0.010                                                                      0.054 ± 0.010                                                                      9.1 ± 2.7                  Liver    0.46 ± 0.08                                                                        0.40 ± 0.16                                                                        0.87 ± 0.18                                                                       0.43 ± 0.03                                                                        0.082 ± 0.010                                                                      4.7 ± 0.3                  Kidney   2.02 ± 0.32                                                                        3.5 ± 1.8                                                                          24.3 ± 1.6                                                                        1.79 ± 0.82                                                                        1.26 ± 0.19                                                                        4.6 ± 0.4                  Muscle   0.044 ± 0.006                                                                      0.029 ± 0.023                                                                      0.13 ± 0.06                                                                       0.028 ± 0.005                                                                      0.037 ± 0.001                                                                      2.04 ± 0.29                Tumor    5.2 ± 1.5                                                                           1.0 ± 0.29                                                                        0.26 ± 0.09                                                                       2.22 ± 0.36                                                                        0.094 ± 0.004                                                                      10.9 ± 0.2                 Tumor mass (g)                                                                         0.21 ± 0.07                                                                        0.20 ± 0.02                                                                        0.11 ± 0.07                                                                       0.22 ± 0.02                                                                        0.13 ± 0.09                                                                        0.20 ± 0.03                Animal mass (g)                                                                        28.1 ± 1.1                                                                         25.6 ± 2.5                                                                         27.9 ± 2.7                                                                        28.3 ± 0.6                                                                         27.8 ± 2.3                                                                         27.6 ± 2.0                 __________________________________________________________________________     *Values shown represent the mean ± standard deviation of data from         three animals (n = 2 for Group 2).                                            .sup.† Group 2 -- Mice on normal (folaterich) diet; Group 3 --         folate receptor blocked with proadministration of folate; Group 4 --          folate chase (i.v.) 3.5 hours following .sup.67 GaDF-Folate                   administration.                                                               .sup.‡ Animals sacrificed 4.5 hours (rather than 4 hours)          following .sup.67 GaDF-Folate administration.                            

                                      TABLE 4                                     __________________________________________________________________________    Tumor to Background Tissue Contrast Obtained with .sup.67 Ga-Radiotracers     in the Athymic Mouse Model                                                            Tumor-to-Non-target Ratio 4 Hours Following Intravenous                       Administration of Radiotracers*                                               .sup.67 Ga-DF-Folate.sup.†                                                                          .sup.67 Ga-DF                                                                        .sup.67 Ga-citrate                        Group 1                                                                              Group 2                                                                              Group 3 Group 4.sup.‡                                                             Group 5                                                                              Group 6                           __________________________________________________________________________    Tumor/blood                                                                           409 ± 195                                                                         60 ± 18                                                                           0.64 ± 0.31                                                                        48 ± 5                                                                            3.9 ± 1.2                                                                         0.84 ± 0.19                    Tumor/muscle                                                                          124 ± 47                                                                          44 ± 24                                                                           2.3 ± 1.2                                                                          82 ± 16                                                                           2.56 ± 0.15                                                                       5.4 ± 0.7                      Tumor/liver                                                                           11.4 ± 3.2                                                                        2.5 ± 0.3                                                                         0.29 ± 0.07                                                                        5.1 ± 0.5                                                                         1.16 ± 0.10                                                                       2.3 ± 0.2                      Tumor/kidney                                                                          2.6 ± 0.9                                                                         0.31 ± 0.08                                                                       0.011 ± 0.004                                                                      1.4 ± 0.5                                                                         0.08 ± 0.01                                                                       2.4 ± 0.3                      __________________________________________________________________________     *Values shown represent the mean ± standard deviation of data from         three animals (n = 2 for Group 2).                                            .sup.† Group 2 -- Mice on normal (folaterich) diet; Group 3 --         folate receptor blocked with proadministration of folate; Group 4 --          folate chase (i.v.) 3.5 hours following .sup.67 GaDF-Folate                   administration.                                                               .sup.‡ Animals sacrificed 4.5 hours (rather than 4 hours)          following .sup.67 GaDF-Folate administration.                            

What is claimed is:
 1. A method for enhancing cellular uptake of adiagnostic agent by a living cell, said method comprising the step ofcontacting the cell with a composition comprising a ligand complex,wherein the ligand complex consists essentially of the diagnostic agentcomplexed with a ligand selected from the group consisting of biotin,biotin receptor-binding analogs of biotin or other biotin-receptorbinding ligands, folate, folate receptor-binding analogs of folate orother folate receptor-binding ligands, riboflavin, riboflavinreceptor-binding analogs of riboflavin or other riboflavinreceptor-binding ligands, and thiamin, thiamin receptor-binding analogsof thiamin or other thiamin receptor-binding ligands, for a timesufficient to permit transmembrane transport of said ligand complex. 2.The method of claim 1 wherein the ligand is folate or folatereceptor-binding analogs of folate.
 3. The method of claim 1 wherein theliving cell is a eukaryote.
 4. The method of claim 3 wherein the livingcell is a human cell.
 5. The method of claim 1 wherein the complex isformed by covalent, ionic or hydrogen bonding of the ligand to thediagnostic agent.
 6. The method of claim 1 wherein the diagnostic agentcomprises a radionuclide.
 7. The method of claim 6 wherein theradionuclide is selected from the group consisting of isotopes ofgallium, indium, copper, technetium, or rhenium.
 8. A method forenhancing cellular uptake of a diagnostic agent by a living cell, saidmethod comprising the step of contacting the cell with a compositioncomprising a ligand complex, wherein the ligand complex consistsessentially of the diagnostic agent complexed through a linker to aligand selected from the group consisting of folate, folaterecepto-binding analogs of folate, or other folate receptor-bindingligands.
 9. The method of claim 8 wherein the complex is formed bycovalent bonding of the ligand to the diagnostic agent.
 10. The methodof claim 8 wherein the linker is a liposome, and the diagnostic agent iscontained in the liposome, said liposome comprising liposome-formingphospholipids, at least a portion of which are covalently bound throughtheir headgroups to the ligand.
 11. The method of claim 8 wherein thediagnostic agent comprises a radionuclide.
 12. The method of claim 8wherein the radionuclide is selected from the group consisting ofisotopes of gallium, indium, copper, technetium, or rhenium.